System for freeze granulation

ABSTRACT

The present invention is a system and method for freeze granulation of a medium such as a biopharmeceutical product. The medium is put into a chamber and mixed using a pair of counter rotating agitators. The agitators have angled blades or paddles on them in order to induce motion in the medium parallel to the axis of the agitator shafts. A set of liquid nitrogen and liquid carbon dioxide nozzles are attached to the chamber. The liquid nitrogen nozzles spray a mist of liquid nitrogen into the medium. The liquid carbon dioxide nozzles spray carbon dioxide snow into the chamber. The liquid nitrogen and the carbon dioxide can be sprayed into the chamber at the same time or alternately.

This application is a continuation of U.S. patent application Ser. No.09/003,288, filed Jan. 6, 1998, now U.S. Pat. No. 5,964,100.

FIELD OF THE INVENTION

The present invention relates to the freezing of products using liquidnitrogen and carbon dioxide snow, and in particular to the creation offrozen granules of the product using both liquid nitrogen and carbondioxide snow.

BACKGROUND

Cryopreservation and cryoprocessing of mediums such asbiopharmeceuticals and foods are important in the manufacturing, use,and sale of these products. However, in order to process many of theseproducts, the cryopreservation or cryoprocessing must be done uniformlyand in a controlled manner or the value of the product may be lost. Forexample, when processing cells for cryopreservation, if the cells arefrozen too quickly with too high of a water content, then the cells willrupture and become unviable.

Uniformity can be achieved using small containers in which the volume ofthe medium in the container is small enough to allow it to be uniformlycooled. However, such small containers only allow small quantities of aproduct to be processed at one time, and thus are of limited commercialvalue. Additionally, as the size of the container is increased, theprocessing speed must be reduced in order to maintain uniformity in theprocessing.

One technique which has been used to cryoprocess foods in largerquantities is a system which mixes the product as it is frozen. Usingtraditional refrigerators or freezers during this process would take toolong since the heat is extracted from the medium too slowly. Therefore,commercial systems have operated by spraying carbon dioxide snow on theproduct while it is mixed in order to rapidly cool it. Carbon dioxidesnow, however, is a solid and therefore often makes less thermallyconductive contact with solids in the medium and will not usually coolthem quickly. This is beneficial for products which can be denatured ifthey are cooled too rapidly.

Liquid nitrogen is colder than carbon dioxide snow and can more quicklyextract heat from a medium or product. Some commercial cryoprocessingsystems have operated by spraying liquid nitrogen on the product ormixing the product with a liquid nitrogen mist. This allows for veryrapid processing of a product and makes liquid nitrogen very useful incommercial cryoprocessing. Liquid nitrogen, however, may cool a producttoo quickly and can damage or destroy the usefulness of the product by,for example, denaturing it.

What is needed is a system and a method for cryoprocessing orcryopreservation of a medium or product which can make use of theproperties of carbon dioxide snow and liquid nitrogen for cryoprocessingor cryopreservation of a medium or product without destroying itscommercial utility.

SUMMARY OF INVENTION

One embodiment of the present invention is a system and method forfreeze granulation of a medium such as a biopharmeceutical product. Themedium is put into a chamber and mixed using a pair of counter rotatingagitators. The agitators have angled blades or paddles on them in orderto induce motion in the medium parallel to the axis of the agitatorshafts. A set of liquid nitrogen and liquid carbon dioxide nozzles areattached to the chamber. The liquid nitrogen nozzles spray a mist ofliquid nitrogen into the medium. The liquid carbon dioxide nozzles spraycarbon dioxide snow into the chamber. The liquid nitrogen and the carbondioxide can be sprayed into the chamber at the same time or alternately.

The liquid nitrogen mist and the carbon dioxide snow contact the mediumwhen introduced into the chamber and the medium freezes. As the mediumfreezes the agitators mix the medium creating frozen granules of themedium.

Another embodiment of the present invention relates to a system forcryoprocessing or cryopresevation of a medium in a chamber. Oneembodiment of the invention comprises a chamber, a mixer, the mixerbeing housed within the chamber and coupled to the chamber. The chamberhas a first input port for inputting carbon dioxide snow into thechamber. The chamber has a second input port for inputting a mist ofliquid nitrogen droplets into the chamber. In other embodiments of theinvention the medium is a biopharmeceutical product, a food product, ora biotechnology product.

In yet another embodiment of the invention, the mixer includes one ormore agitators. In another embodiment of the invention, the agitatorsinclude a pair of counter rotating agitators. In other embodiments ofthe invention the mixer moving the material which first comes intocontact with the carbon dioxide snow after the carbon dioxide snow isintroduced into the chamber.

In still other embodiments of the invention, the mixer is a variablespeed mixer, operates at different speeds, or with different agitators.In yet other embodiments of the invention the mixer is configured tocreate a flow of the medium about a perimeter of the chamber. In otherembodiments of the invention second input port contains a nozzle whichis configured to generate the liquid nitrogen mist wherein an averagediameter of drops in the liquid nitrogen mist is less than 5 microns, 20microns, 100 microns, or 1 millimeter.

In still other embodiments of the invention the carbon dioxide snow orthe liquid nitrogen mist is input into the chamber at a rate which iscontrolled based on a feedback loop which monitors information includinga load on the agitator, a temperature of the medium, or the temperatureof the exhaust from the chamber. In still other embodiment of theinvention a feedback loop controls a protocol by which the medium is. Inother embodiments of the invention the chamber is connected to abioreactor chamber, a processor, a feeder, a filtration system, or areactor.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts a side view of one embodiment of the invention showingone of the agitators and four representative paddles extending from theaxis of the agitator. Additionally, liquid carbon dioxide valves, carbondioxide snow nozzles, and liquid nitrogen nozzles are depicted.

FIG. 2 is a front view of an embodiment of the invention showing a pairof counter rotating agitators and an optional heating jacket.

FIG. 3 is a top view of an embodiment of the invention showing a globalcirculation pattern induced in the medium by the angling of the paddleson the agitators.

FIG. 4A depicts the angling of the paddles on an agitator to induce flowof the medium in a direction along the axis of the agitator.Additionally, the angling of the paddles induces an increase in thelevel of the medium in the direction of flow of the medium.

FIG. 4B depicts the level of the medium induced on both sides of thechamber through the action of both agitators.

FIG. 5 depicts a detailed sketch of an embodiment of the inventionshowing a scrapper blade.

FIG. 6A is a graph showing one possible cooling protocol for anembodiment of the invention.

FIG. 6B is a graph showing power consumption for the agitator motors atconstant agitation speed versus time for a particular medium as it iscooled following the protocol depicted in graph 6A.

FIG. 7 is a graph depicting another protocol in which there are twodifferent cooling rates Q1 and Q2.

FIG. 8 shows one embodiment of a system containing many subcomponentsfor processing a medium using a freeze granulator

FIG. 9 depicts 3 configurations which can be used to allow a freezegranulator chamber to be emptied.

FIG. 10 depicts another embodiment of the invention with a single rotorconfiguration.

DETAILED DESCRIPTION

FIGS. 1 and 2 depict a side view and a front view, respectively, of oneembodiment of the present invention. FIG. 1 shows granulating freezersystem 100. Granulating freezer system 100 includes chamber 102. Chamber102 can be of any shape or configuration and is depicted as rectangularfor illustrative purposes only. Chamber 102 should be made of a materialwhich can withstand prolonged contact with the medium to be treated orfrozen in the chamber.

Inside chamber 102 is a pair of counter rotating agitators 104. In theside view of FIG. 1, only one of the agitators is visible. FIG. 2 showsa front view in which the pair of counter rotating agitators 104 areshown rotating towards each other as depicted by arrows 106. Each of theagitators includes one or more paddles 108. Paddles 108 can be in anyshape or form which will cause movement of the medium. For examplepaddles 108 can be of any desired cross section and need not all be thesame shape, the same size, or uniformly distributed along the axis ofthe agitator. Additionally, paddles 108 can be one or more continuouscurves such as a cork screw shape around shaft 110 of agitator 104.

Paddles 108 mix the medium so that it is uniformly cooled. Paddles 108create movement of the medium in the direction of arrows 106 in FIG. 2.Additionally, in another embodiment of the invention, paddles 108 areangled so that circulation of the medium is created in the direction ofarrow 112 in FIG. 1. FIG. 3 shows a top view of chamber 102. The angledpaddles create circulation of the medium about a perimeter of chamber102 as depicted by arrow 114. This global circulation helps ensureuniformity of the medium throughout chamber 108.

In one embodiment of the invention, the angle of the paddles is variedas a function of the distance of the paddle from an end of the chamber.As depicted in FIG. 4A, paddles 402A through 402G are increasinglyangled to create a faster flow and a higher level of the medium at endof chamber 404. The paddles for the agitator on the other side of thechamber are angled to create a faster flow and higher level of themedium in the opposite direction. This is depicted in FIG. 4B. Thebuildup of material at alternate ends of the chamber aids the creationof a circulation pattern like the one indicated by arrow 114 in FIG. 3.

In another embodiment of the invention not depicted in the figures, thepaddles are angled so that two independent circulation patterns arecreated instead of the one shown in FIG. 3. In other embodimentsmultiple circulation patterns can be created. Any circulation patternwhich will help to mix the contents of the chamber can be used.

In the embodiment of the invention depicted in FIG. 1, optional scrapperblade 115 is used to prevent a buildup of the medium at the ends ofchamber 102. FIG. 5 shows a detail sketch of a scrapper blade 515.Scrapper blade 515 can be configured so that it touches the end of thechamber or so that there is a gap between the scrapper blade and the endof the chamber. The gap can be 1 millimeter, one-sixteenth of an inch,one eighth of an inch, one fourth of an inch, one half of an inch, orlarger than one half of an inch.

FIG. 2 depicts an embodiment of the invention in which optional heatinglayer 202 covers a portion of chamber 102. In this embodiment of theinvention, a portion of the exterior of chamber 102 is insulated orslightly heated to generate a thin liquid film of the medium along theinner surface of chamber 102. Heating layer 202 can be maintained at atemperature just above the freezing point of the medium in the containerso that even when the medium is becoming viscous or forming solidgranules, there is a liquid layer along the inner walls of chamber 102.The liquid layer can help prevent the medium from sticking to the wallsor piling up along a portion of chamber 102. Additionally, the liquidfilm can act as a lubricating layer, reducing the resistance of themedium on the agitators. The heating of heating layer 202 can beachieved using any conventional heat transfer mechanism including butnot limited to water or other fluids such as silicon or oil, electricheaters, or infrared radiation.

In FIG. 1 liquid carbon dioxide is input into chamber 102 through valves116 which are connected to nozzles 118. When the liquid carbon dioxideat high pressure passes through valve 116 and into nozzle 118, thepressure drops and the liquid carbon dioxide turns into solid carbondioxide snow. The particles of carbon dioxide snow fall onto the mediumand are then mixed in by agitators 104. Liquid nitrogen mist is inputinto chamber 102 through valves 120 which are connected to nozzles 122.The liquid nitrogen mist is deposited on the medium and mixed in byagitators 104.

In one aspect of the invention, paddles 108 extend above the surface ofthe medium in chamber 102 during some portion of their rotation cycle.When paddles 108 extend above the surface, they contact material on thesurface, including carbon dioxide snow which may have accumulated on thesurface. The surface layer, including the carbon dioxide snow is forceddown into the medium by paddles 108. In other embodiments of invention,the paddles extend above the surface of the medium by less than 1 inch,less than 2 inches, more than 2 inches, 5%, 20%, or 95% of the diameterof circle made by the tip of rotating paddles 108.

When the present invention is used to cool or freeze the medium, thesize of the liquid nitrogen drops used must be controlled. Liquidnitrogen is typically at a temperature much lower than that of solidcarbon dioxide. The extreme cold of the liquid nitrogen mist drops, whenthey come in contact with the medium, may cause damage to the medium.For example, if the medium is a biopharmeceutical product it can bedamaged if it is frozen while containing too much excess water. Liquidnitrogen may cause granules of the medium to freeze too rapidly withoutallowing sufficient time for excess water to be released from theproduct. As a result, frozen water in the granule may cause damage tothe biopharmeceutical product.

In one embodiment of the invention the liquid nitrogen nozzle isconfigured to produce liquid nitrogen drops at or below a specifiedaverage size. Controlling the size of the drops and the rate at whichthe drops are input into the chamber allows for control of the coolingrate of the medium. In other aspects of the invention the liquidnitrogen nozzle is configured to produce drops with an average diameterof less than 5 microns, about 5 microns, less than 20 microns, about 20microns, less than 100 microns, about 100 microns, less than 1millimeter, or about 1 millimeter.

In the embodiment of the invention depicted in FIG. 1, chamber 102includes baffle 124. Baffle 124 is placed in chamber 102 to deflectcarbon dioxide snow particle. Baffle 124 may be positioned to ensure adirected or uniform deposition of carbon dioxide snow within chamber102. Additionally, baffles 124 can be positioned to prevent carbondioxide snow particles from being withdrawn from chamber 102 through gasexhaust 126 before the carbon dioxide snow particles have contacted themedium in the chamber. Gas exhaust 126 removes excess gas from chamber102 to regulate the pressure inside chamber 102. Without baffles 124, acurrent set up by exhaust 126 would remove some of the carbon dioxidesnow from chamber 102 before it contacted the surface of the medium.This would reduce the effectiveness of freeze granulation system 100.

In another embodiment of the invention, the rate at which carbon dioxidesnow, and liquid nitrogen mist are introduced into the chamber iscontrolled by a computer. Either or both of these rates can be computercontrolled along with the size of the drops in the liquid nitrogen mist,the speed of the agitators, the angles of the paddles, and the rate ofmixing of the medium. Any or all of these parameters can be computercontrolled based on a feedback loop which monitors variables in thegranulation processing. Parameters which can be used by the computer tofeedback on, include but are not limited to, the temperature of themedium, the temperature uniformity of the medium, temperature gradientsin the medium, the temperature of the chamber, the temperature ofexhaust gases, ratios or differences of any measured temperatures, theload on the agitators, the power consumption of the agitators, and/orthe speed of the agitators.

In another embodiment of the invention, parameters, including thoselisted above can be controlled over time to achieve desired processingprotocols. As depicted in the graphs in FIG. 6, the temperature of themedium over time can be controlled as desired to achieve particularprocessing characteristics. Graph 6A depicts a temperature versus timeprofile in which a liquid is cooled to 0 degrees Celsius and held atthat temperature for a specified period of time. At the end of the holdperiod, the medium is cooled at a rate of Q degrees per second untiltemperature T is reached. This protocol may be useful in freezegranulating biopharmeceutical products so that excess water can bewithdrawn from the product during the temperature plateau at 0 degreesCelsius.

Graph 6B depicts power consumption for the agitator motors as a functionof time for the case in which the medium is a suspension of cells. Whenthe medium is cooled to 0 degrees Celsius it is initially a liquid. Asthe medium is held at 0 degrees Celsius, water is removed from the cellsand ice crystals form in the slurry surrounding the cells increasing theviscosity of the medium. After the medium has been held at 0 degreesCelsius for a time t1 the medium becomes more viscous and creates moreresistance for the agitators for a constant agitation speed. Thisresults in a slight rise in the power requirements for the agitators.After a time t2 the medium achieves a slightly higher viscosity and thepower requirements for the agitators increase slightly more. Finally, attime t4 the viscosity peaks as frozen granules are formed. At thispoint, the feedback system can begin to decrease the temperature below 0degrees Celsius since the medium has solidified into granules. This isdepicted in FIG. 6A by the downward slope of the temperature versus timecurve. FIG. 6B is an example of a parameter, power consumption, that maybe used by a computer controlled feedback system to achieve a desiredprotocol. FIG. 6B shows how once granule formation has been completed,the power consumption of the agitators falls dramatically. Otherparameters can be used by the feedback system without departing from thepresent invention.

In general, as the consistency of a medium changes from a liquid to aviscous mass just prior to breaking up into granules, the power requiredto agitate or mix the medium increases. The peak power requirements forthe agitators can be reduced through the use of a heating jacket such asthe one depicted in FIG. 2. The thin layer of liquid created by theheating jacket reduces the viscosity of the medium near the walls of thecontainer, reducing the power needed to nix the medium. The jacket canbe heated just prior to the peak power points or continuously throughoutthe cooling process. Once the medium breaks up into granules, itsviscosity decreases rapidly and the power required to mix it is reduceddramatically.

FIG. 7 shows another example of a protocol of temperature versus timewhich can be achieved using the present invention. The medium is cooledto 0 degrees Celsius and held there for a time t1. The medium is thencooled at a rate of Q1 degrees per second to a temperature T1 and heldthere for t2 seconds. The medium is then cooled at a rate of Q2 degreesper second to a temperature of T2 and held there until needed.Additionally, processing is possible using the present invention. Asdepicted by the dotted curve at the T1 temperature plateau, thetemperature versus time profile can be varied in a non-linear fashion ifdesired.

FIG. 8 depicts an embodiment of a freeze granulator system includingmany processing subcomponents. In this embodiment of the invention,bioreactor chamber 802 is coupled to centrifuge 804, filtration system806, and granulation chamber 808 through a system of pipes and valves.Bioreactor chamber 802, centrifuge 804 and filtration system 806 allow amedium, such as a biopharmeceutical product, or a protein to beprocessed prior to freeze granulation in granulation chamber 808. Otherprocessing systems can be attached to granulation chamber 808 withoutdeparting from the present invention, including but not limited to foodprocessing equipment, cosmetics processing equipment, or biotechnologyprocessing equipment.

Freeze granulator 808 is coupled to liquid nitrogen storage container810 and liquid carbon dioxide storage container 812. Liquid nitrogenmist and carbon dioxide snow are input into granulation chamber 808through values 814 and 816 respectively. In other embodiments of theinvention, storage containers 810 and 812 include pressurizing cellswhich maintain the pressure inside containers 810 and 812 above aminimum level. This may be especially important for liquid carbondioxide container 812 because liquid carbon dioxide may solidify if notkept at high enough pressure.

Exhaust gases are vented from freeze granulator 808 through exhaust line818. Exhaust line 818 is connected to the top of freeze granulator 808,and to particle separator 820. Particle separator 820 is connected toheater 822 and gas filters 824. Particle separator 820 removes largeparticles from the exhaust gas before the gas gets to filter 824. Heater822 is used to warm the exhaust gas to prevent solid particles such ascarbon dioxide snow particles from clogging filter 824. Exhaust blower826 pulls the exhaust gas through the exhaust line and pushes it outthrough gas out line 828. In other embodiments of the invention filtersets can be used instead of filter 824. one filter at a time from theset can be automatically switched into position which the other filtersin the set are cleaned. This allows a clean filter to continuously bepositioned in the exhaust line. It is noted that other embodiments ofthe invention do not include particle separator 820, heater 822, gasfilters 824, exhaust blower 826 and/or gas out line 828.

After the medium has been freeze granulated, it is emptied out of freezegranulator 808 through output 830 and can be stored or transferred toanother site or container. In one embodiment of the invention, filterand blower 832 is used when the medium is emptied from freeze granulator808 in order to maintain a positive pressure inside freeze granulator808.

In an embodiment of the invention not depicted in FIG. 9, the seal forthe agitator shafts extending into freeze granulator 808 are kept at apressure higher than that in freeze granulator 808 using, for example,filtered dry gas. The increased pressure in the seals prevents thecontents of freeze granulator 808 from coming into contact with the sealsurfaces. Additionally, pressurizing the seals can be used to detectpossible seal failures. If the seals are pressurized and the seals fail,leaking into the chamber, then the pressure in the chamber willincrease. Additionally, pressure sensors connected to the seals willdetect a drop in pressure if the seals fail.

In another embodiment freeze granulator 808 is coupled to a cleaningsolution inlet. When it is desired to clean freeze granulator 808, it ispartially filled with cleaning solution, and the agitators are turnedon. The agitators can be run at high speed to ensure a thorough cleaningof freeze granulator 808, and the cleaning fluid may be recirculatedthrough the seal chambers in order to clean them. A heating layer arounda portion of freeze granulator 808, if present, may be used duringcleaning to heat the cleaning fluid, and temperature monitors can beused to monitor and control the cleaning process.

In another embodiment of the invention, steam or hydrogen peroxide vapormay be used to sterilize the system. A heating layer, if present, may beused to heat the freeze granulator and aid in the steam or hydrogenperoxide vapor cleaning.

Referring to FIG. 8, computer controller 832 couples temperatureindicator and controller 834, agitator motor monitor and controller 836,liquid nitrogen valves 814, liquid carbon dioxide valves 816, andexhaust gas line temperature and pressure monitor 838 in a closedfeedback system. The temperature of the medium in the freeze granulator808 and the exhaust gas are measured using sensors 834 and 838respectively. The load and speed of the agitators is measured usingsensor 836. These measured values are used to feedback through computercontroller 832 in order to adjust the flow rate of liquid nitrogen andcarbon dioxide snow into the freeze granulator 808 using valves 814 and816 respectively.

Using this feedback loop a predetermined protocol can be achieved. Forexample, the medium in freeze granulator 808 can be maintained at aconstant temperature for a specified period of time by measuring thetemperature of the medium using sensor 834 and maintaining thetemperature at a constant level by varying the flow of liquid nitrogeninto freeze granulator 808 through valve 814 and/or varying the flow ofcarbon dioxide snow into freeze granulator 808 using valve 816. In orderto achieve this result, a control signal is generated by computercontroller 832 which is proportional to the difference between theactual temperature measured by sensor 834 and a desired value. Thecontrol signal is used to adjust the actuators connected to valves 814and 816 in order to allow more or less liquid nitrogen or carbon dioxidesnow into freeze granulator 808.

It is noted that more than one temperature sensor can be used withoutdeparting from the present invention. Additionally, computer controller832 could alternatively be implemented using any analog or digitalcontrol system.

FIG. 9 shows three alternative embodiments for freeze granulator 901which can be used to discharge the medium. In FIG. 9A central plug 902between agitators 904 is lifted opening up chute 906 to allow the mediumout of freeze granulator 901. In FIG. 9B central plug 908 betweenagitators 910 drops down to allow the medium to exit freeze granulator912 through chute 914. In FIG. 9C, bottom section 916 of freezegranulator 918 is hinged at hinge 920 and can be opened to position 922to empty the medium from freeze granulator 918.

FIG. 10 depicts another embodiment of the invention. In this embodimentchamber 1002 has inputs for liquid nitrogen 1004 and carbon dioxide snow1006 at the top, and single rotor 1008 at the bottom. This configurationis useful for small volumes.

What is claimed is:
 1. A method for cryopreservation of a medium in achamber, comprising: introducing a carbon dioxide snow into the chamber;introducing a liquid nitrogen mist into the chamber at the same time thecarbon dioxide snow is introduced into the chamber; and mixing themedium until it is cryopreserved.
 2. The method of claim 1, wherein: themedium is a biopharmaceutical product.
 3. The method of claim 1,wherein: the medium is a food product.
 4. The method of claim 1,wherein: the medium is a biotechnology product.
 5. The method of claim1, wherein: mixing the medium includes mixing the medium with counterrotating agitators.
 6. The method of claim 5, wherein: the agitatorsincludes a paddle.
 7. The method of claim 6, wherein: a mixing surfaceof the paddle is at angle greater than 90 degrees to the direction ofmotion of the paddle.
 8. The method of claim 6, wherein: a mixingsurface of the paddle is at about an angle of 90 degrees to thedirection of motion of the paddle.
 9. The method of claim 5, wherein:the agitators includes a plurality of paddles.
 10. The method of claim5, wherein: mixing includes varying the speed of the agitators.
 11. Themethod of claim 5, wherein: mixing includes operating the agitators atdifferent speeds.
 12. The method of claim 5, wherein: mixing the mediumincludes creating a flow of the medium about a perimeter of the chamber.13. The method of claim 12, wherein: mixing the medium includes creatinga flow of the medium transverse to the flow of the medium about theperimeter of the chamber.
 14. The method of claim 12, wherein: theperimeter of the chamber includes a segment parallel to the axis ofrotation of at least one of the agitators.
 15. The method of claim 5,wherein: the chamber includes a structure to allow for thermally inducedchanges in size of the chamber.
 16. The method of claim 15, wherein: thestructure includes an expansion bearing attached to at least one of theagitators.
 17. The method of claim 1, wherein: mixing the mediumincludes moving the material which first comes into contact with thecarbon dioxide snow after the introducing of the carbon dioxide snowinto the chamber.
 18. The method of claim 1, wherein: mixing includesvarying the rate of mixing.
 19. The method of claim 1, comprising:introducing the carbon dioxide snow into the chamber from a pressurizedcarbon dioxide container.
 20. The method of claim 19, comprising:maintaining a predetermined minimum pressure in the pressurized carbondioxide container.
 21. The method of claim 1, wherein: an averagediameter of drops in the liquid nitrogen mist is about 5 microns. 22.The method of claim 1, wherein: an average diameter of drops in theliquid nitrogen mist is less than 5 microns.
 23. The method of claim 1,wherein: an average diameter of drops in the liquid nitrogen mist isabout 20 microns.
 24. The method of claim 1, wherein: an averagediameter of drops in the liquid nitrogen mist is less than 20 microns.25. The method of claim 1, wherein: an average diameter of drops in theliquid nitrogen mist is about 100 microns.
 26. The method of claim 1,wherein: an average diameter of drops in the liquid nitrogen mist isless than 100 microns.
 27. The method of claim 1, wherein: an averagediameter of drops in the liquid nitrogen mist is about 1 millimeter. 28.The method of claim 1, wherein: an average diameter of drops in theliquid nitrogen mist is less than 1 millimeter.
 29. The method of claim1, wherein: the rate of introducing of carbon dioxide snow into thechamber is controlled based on a feedback loop which monitorsinformation including a temperature of the medium.
 30. The method ofclaim 1, wherein: mixing includes mixing using an agitator; and the rateof introducing of carbon dioxide snow into the chamber is controlledbased on a feedback loop which monitors information including a load onthe agitator.
 31. The method of claim 1, wherein: mixing includes mixingusing an agitator; and the rate of introducing of carbon dioxide snowinto the chamber is controlled based on a feedback loop which monitorsinformation including a temperature of the medium and a load on theagitator.
 32. The method of claim 1, wherein: the rate of introducing ofliquid nitrogen mist into the chamber is controlled based on a feedbackloop which monitors information including a temperature of the medium.33. The method of claim 32, wherein: a temperature of the medium ismeasured at more than one location.
 34. The method of claim 1, wherein:mixing includes mixing using an agitator; and the rate of introducing ofliquid nitrogen mist into the chamber is controlled based on a feedbackloop which monitors information including a load on the agitator. 35.The method of claim 1, wherein: mixing includes mixing using anagitator; and the rate of introducing of liquid nitrogen mist into thechamber is controlled based on a feedback loop which monitorsinformation including a temperature of the medium and a load on at theagitator.
 36. The method of claim 1, wherein: mixing includes mixingusing an agitator; and the rate of introducing of carbon dioxide snowinto the chamber is controlled based on a feedback loop which monitorsinformation including a temperature of the medium and a load on theagitator.
 37. The method of claim 1, wherein: the rate of introducing ofliquid nitrogen mist into the chamber is controlled by a computercontrolled feedback loop.
 38. The method of claim 1, wherein: the rateof introducing of carbon dioxide snow into the chamber is controlled bya computer controlled feedback loop.
 39. The method of claim 1, wherein:the rate of introducing of carbon dioxide snow and liquid nitrogen mistinto the chamber is controlled by a computer controlled feedback loop.40. The method of claim 1, wherein: the rate of mixing of the medium inthe chamber is controlled by a computer controlled feedback loop. 41.The method of claim 1, wherein: the rate of mixing of the medium in thechamber and the rate of introducing of carbon dioxide snow and liquidnitrogen mist into the chamber is controlled by a computer controlledfeedback loop.
 42. The method of claim 41, wherein: the computercontrolled feedback loop measure parameters including a temperature ofthe medium in the chamber and a load on at least one of the agitators.43. The method of claim 1, wherein: the medium is cooled at apredetermined rate.
 44. The method of claim 1, wherein: the medium iscooled following a predetermined protocol.
 45. The method of claim 44,wherein: the predetermined protocol includes varying the rate at whichthe medium is cooled.
 46. The method of claim 44, wherein: thepredetermined protocol includes varying a rate of the mixing.
 47. Themethod of claim 1, comprising: a bioreactor chamber connected to thechamber.
 48. The method of claim 47, comprising: a filtration systemconnected to the bioreactor chamber and to the chamber.
 49. The methodof claim 1, comprising: a reactor chamber connected to the chamber. 50.The method of claim 1, comprising: a processor connected to the chamber.51. The method of claim 1, comprising: a feeder connected to thechamber.
 52. The method of claim 1, wherein: the carbon dioxide snow isintroduced into the chamber using a valve which is capable of shuttingoff the flow of carbon dioxide snow into the chamber and then restartingthe flow of carbon dioxide snow into the chamber.
 53. A method forcryopreservation of a medium in a chamber, comprising: stirring themedium; introducing carbon dioxide snow into the chamber using a liquidcarbon dioxide valve wherein the liquid carbon dioxide valve is capableof capable of shutting off the flow of carbon dioxide snow into thechamber and then restarting the flow of carbon dioxide snow into thechamber; and introducing liquid nitrogen mist into the chamber after themedium has started to solidify, and continuing the introducing until themedium is cryopreserved.
 54. The method of claim 53, wherein: the mediumis a biopharmaceutical product.
 55. The method of claim 53, wherein: themedium is stirred using counter rotating agitators.
 56. The method claim53, wherein: the rate at which the carbon dioxide snow is introducedinto the chamber is determined by information including the amount offorce required to mix the medium in the chamber.